ABSTRACT Despite decades of advances in clinical care, the high incidence of staphylococcal infections remain a major public health concern. Multiple antibiotic-resistant isolates of Staphylococcus aureus and coagulase-negative Staphylococcus epidermidis are frequently isolated in the hospital environment and significantly limit therapeutic options. Indeed, it is increasingly becoming evident that a fundamental understanding of staphylococcal biology and factors affecting its fitness are required for the development of effective therapeutics. Towards this goal, we have recently identified important physiological functions for arginine catabolic enzymes including nitric oxide synthase (NOS) and arginine deiminase (ADI), in promoting growth and controlling programmed cell death (PCD) in staphylococci. Under normal physiological conditions, NOS activity promotes growth by activating respiration and preventing metabolic dysfunction including excessive production of the polysaccharide intracellular adhesin (PIA). However, influx of acetate (a byproduct of glucose metabolism) into the cytoplasm under acidic conditions appears to corrupt normal function of NOS and promotes NOS-dependent production of reactive oxygen species (ROS) and PCD. Interestingly, carbon flux through the ADI pathway appears to counter NOS-dependent PCD. The mechanism by which these pathways affect staphylococcal physiology is still under intense investigation and constitutes the subject of this application. In Aim #1, we will test how NOS promotes growth by suppressing PIA production as part of its normal physiological activity. Further, we will investigate if phenylalanine plays a role in NOS-dependent PIA suppression and whether NOS activity affects the pathogenic potential of staphylococci using an infant mouse model of central nervous system (CNS) catheter infection. In Aim #2, we will test the hypothesis that NOS-dependent ROS production during PCD results from NOS uncoupling, a phenomenon wherein NOS generates superoxide rather than nitric oxide as byproduct. Finally in Aim #3, we will test the potential for the arginine deiminase and the arginase pathways in countering PCD by activating catalase activity through glutamate biosynthesis. On completion of these studies, we will gain a deeper mechanistic understanding of how various arginine catabolic pathways affect staphylococcal physiology and fitness.